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April 21, 2020  |  

Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles: A GeT-RM Collaborative Project.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.Published by Elsevier Inc.

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April 21, 2020  |  

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time

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April 21, 2020  |  

Evolution of a 72-kb cointegrant, conjugative multiresistance plasmid from early community-associated methicillin-resistant Staphylococcus aureus isolates.

Horizontal transfer of plasmids encoding antimicrobial-resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s the first CA-MRSA isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline and penicillin-resistance genes on plasmid pWBG753 (~30 kb). WA-5 and pWBG753 appeared only briefly in WA, however, fusidic-acid-resistance plasmids related to pWBG753 were also present in the first European CA-MRSA at the time. Here we characterized a 72-kb conjugative plasmid pWBG731 present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749-family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium and penicillin-resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs) and the BinL resolution system of the ß-lactamase transposon Tn552 An evolutionary intermediate ~42-kb non-conjugative plasmid pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline-resistance plasmid pT181. IS257 likely facilitated replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized non-conjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous CA-MSSA. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.Copyright © 2019 American Society for Microbiology.

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April 21, 2020  |  

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.

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April 21, 2020  |  

Whole genome sequence of first Candida auris strain, isolated in Russia.

Candida auris is an emergent yeast pathogen, easily transmissible between patients and with high percent of multidrug resistant strains. Here we present a draft genome sequence of the first known Russian strain of C. auris, isolated from a case of candidemia. The strain clustered within South Asian C. auris clade and seemingly represented an independent event of dissemination from the original species range. Observed fluconazole resistance was probably due to F105L and K143R mutations in ERG11. © The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.

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April 21, 2020  |  

Insights into the bacterial species and communities of a full-scale anaerobic/anoxic/oxic wastewater treatment plant by using third-generation sequencing.

For the first time, full-length 16S rRNA sequencing method was applied to disclose the bacterial species and communities of a full-scale wastewater treatment plant using an anaerobic/anoxic/oxic (A/A/O) process in Wuhan, China. The compositions of the bacteria at phylum and class levels in the activated sludge were similar to which revealed by Illumina Miseq sequencing. At genus and species levels, third-generation sequencing showed great merits and accuracy. Typical functional taxa classified to ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), denitrifying bacteria (DB), anaerobic ammonium oxidation bacteria (ANAMMOXB) and polyphosphate-accumulating organisms (PAOs) were presented, which were Nitrosomonas (1.11%), Nitrospira (3.56%), Pseudomonas (3.88%), Planctomycetes (13.80%), Comamonadaceae (1.83%), respectively. Pseudomonas (3.88%) and Nitrospira (3.56%) were the most predominating two genera, mainly containing Pseudomonas extremaustralis (1.69%), Nitrospira defluvii (3.13%), respectively. Bacteria regarding to nitrogen and phosphorus removal at species level were put forward. The predicted functions proved that the A/A/O process was efficient regarding nitrogen and organics removal. Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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April 21, 2020  |  

Complete genome of Pseudomonas sp. DMSP-1 isolated from the Arctic seawater of Kongsfjorden, Svalbard

The genus Pseudomonas is highly metabolically diverse and has colonized a wide range of ecological niches. The strain Pseudomonas sp. DMSP-1 was isolated from Arctic seawater (Kongsfjorden, Svalbard) using dimethylsulfoniopropionate (DMSP) as the sole carbon source. To better understand its role in the Arctic coastal ecosystem, the genome of Pseudomonas sp. strain DMSP-1 was completely sequenced. The genome contained a circular chromosome of 6,282,445?bp with an average GC content of 60.01?mol%. A total of 5510 protein coding genes, 70 tRNA genes and 19 rRNA genes were obtained. However, no genes encoding known enzymes associated with DMSP catabolism were identified in the genome, suggesting that novel DMSP degradation genes might exist in Pseudomonas sp. strain DMSP-1.

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April 21, 2020  |  

Extended haplotype phasing of de novo genome assemblies with FALCON-Phase

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy was affected by heterozygosity of the individual sequenced, with higher accuracy for cattle and zebra finch (>97%) compared to human (82%). In addition, scaffolding with the same Hi-C chromatin contact data resulted in phased chromosome-scale scaffolds.

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April 21, 2020  |  

Complete genome sequence of Bacillus velezensis JT3-1, a microbial germicide isolated from yak feces

Bacillus velezensis JT3-1 is a probiotic strain isolated from feces of the domestic yak (Bos grunniens) in the Gansu province of China. It has strong antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Mannheimia haemolytica, Staphylococcus hominis, Clostridium perfringens, and Mycoplasma bovis. These properties have made the JT3-1 strain the focus of commercial interest. In this study, we describe the complete genome sequence of JT3-1, with a genome size of 3,929,799 bp, 3761 encoded genes and an average GC content of 46.50%. Whole genome sequencing of Bacillus velezensis JT3-1 will lay a good foundation for elucidation of the mechanisms of its antimicrobial activity, and for its future application.

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April 21, 2020  |  

Lateral transfers of large DNA fragments spread functional genes among grasses.

A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism’s adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grass Alloteropsis semialata, a species surmised to exhibit two LGTs, and screen it for other grass-to-grass LGTs using genomic data from 146 other grass species. Through stringent phylogenomic analyses, we discovered 57 additional LGTs in the A. semialata nuclear genome, involving at least nine different donor species. The LGTs are clustered in 23 laterally acquired genomic fragments that are up to 170 kb long and have accumulated during the diversification of Alloteropsis. The majority of the 59 LGTs in A. semialata are expressed, and we show that they have added functions to the recipient genome. Functional LGTs were further detected in the genomes of five other grass species, demonstrating that this process is likely widespread in this globally important group of plants. LGT therefore appears to represent a potent evolutionary force capable of spreading functional genes among distantly related grass species. Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020  |  

The landscape of SNCA transcripts across synucleinopathies: New insights from long reads sequencing analysis

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3textquoteright UTR lengths, as well as novel 5textquoteright starts and 3textquoteright ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114kb SNCA region, with the longest phased block excedding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.

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April 21, 2020  |  

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies

Background New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from textquoteleftfinishedtextquoteright. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.Results We employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: six with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and three with new assemblies based on re-scaffolding or Pacific Biosciences long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: seven for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further seven with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.Conclusions Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our comparisons show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.ADADSEQAGOAGOUTI-basedAGOUTIannotated genome optimization using transcriptome information toolALNalignment-basedCAMSAcomparative analysis and merging of scaffold assemblies toolDPdynamic programmingFISHfluorescence in situ hybridizationGAGOS-ASMGOS-ASMGene order scaffold assemblerKbpkilobasepairsMbpmegabasepairsOSORTHOSTITCHPacBioPacific BiosciencesPBPacBio-basedPHYphysical-mapping-basedRNAseqRNA sequencingQTLquantitative trait lociSYNsynteny-based.

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April 21, 2020  |  

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed.

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double-low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double-low and double-high), accompanied by an increase in genetic load in the double-low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra- and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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April 21, 2020  |  

Large-scale ruminant genome sequencing provides insights into their evolution and distinct traits.

The ruminants are one of the most successful mammalian lineages, exhibiting morphological and habitat diversity and containing several key livestock species. To better understand their evolution, we generated and analyzed de novo assembled genomes of 44 ruminant species, representing all six Ruminantia families. We used these genomes to create a time-calibrated phylogeny to resolve topological controversies, overcoming the challenges of incomplete lineage sorting. Population dynamic analyses show that population declines commenced between 100,000 and 50,000 years ago, which is concomitant with expansion in human populations. We also reveal genes and regulatory elements that possibly contribute to the evolution of the digestive system, cranial appendages, immune system, metabolism, body size, cursorial locomotion, and dentition of the ruminants. Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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